RESUMO
Staphylococcus aureus is a major human pathogen with well-characterized bacteriophage contributions to its virulence potential. Recently, we identified plasmidial and episomal prophages in S. aureus strains using an extra-chromosomal DNA (exDNA) isolation and sequencing approach, uncovering the plasmidial phage ÏBU01, which was found to encode important virulence determinants. Here, we expanded our extra-chromosomal sequencing of S. aureus, selecting 15 diverse clinical isolates with known chromosomal sequences for exDNA isolation and next-generation sequencing. We uncovered the presence of additional episomal prophages in 5 of 15 samples, but did not identify any plasmidial prophages. exDNA isolation was found to enrich for circular prophage elements, and qPCR characterization of the strains revealed that such prophage enrichment is detectable only in exDNA samples and would likely be missed in whole-genome DNA preparations (e.g., detection of episomal prophages did not correlate with higher prophage excision rates nor higher excised prophage copy numbers in qPCR experiments using whole-genome DNA). In S. aureus MSSA476, we found that enrichment and excision of the prophage ÏSa4ms into the cytoplasm was temporal and that episomal prophage localization did not appear to be a precursor to lytic cycle replication, suggesting ÏSa4ms excision into the cytoplasm may be part of a novel lysogenic switch. For example, we show that ÏSa4ms excision alters the promoter and transcription of htrA2 , encoding a stress-response serine protease, and that alternative promotion of htrA2 confers increased heat-stress survival in S. aureus COL. Overall, exDNA isolation and focused sequencing may offer a more complete genomic picture for bacterial pathogens, offering insights into important chromosomal dynamics likely missed with whole-genome DNA-based approaches.
RESUMO
Staphylococcus aureus is a major clinically important pathogen with well-studied phage contributions to its virulence potential. In this commentary, we describe our method to enrich and sequence stealth extra-chromosomal DNA elements in the bacterial cell, allowing the identification of novel extra-chromosomal prophages in S. aureus clinical strains. Extra-chromosomal sequencing is a useful and broadly applicable tool to study bacterial genomics, giving a temporal glance at the extra-chromosomal compartment of the cell and allowing researchers to uncover lower-copy plasmidial elements (e.g., prophages) as well as gain a greater understanding of mobile genetic elements that shuffle on and off the chromosome. Here, we describe how episomal and plasmidial DNA elements can have profound downstream effects on the host cell and surrounding bacterial population, and discuss specific examples of their importance in Gram-positive bacteria. We also offer potential avenues of future research where extra-chromosomal sequencing may play a key role in our understanding of the complete virulence potential of infectious bacteria.
RESUMO
In Staphylococcus aureus, the disease impact of chromosomally integrated prophages on virulence is well described. However, the existence of extra-chromosomal prophages, both plasmidial and episomal, remains obscure. Despite the recent explosion in bacterial and bacteriophage genomic sequencing, studies have failed to specifically focus on extra-chromosomal elements. We selectively enriched and sequenced extra-chromosomal DNA from S. aureus isolates using Roche-454 technology and uncovered evidence for the widespread distribution of multiple extra-chromosomal prophages (ExPΦs) throughout both antibiotic-sensitive and -resistant strains. We completely sequenced one such element comprised of a 43.8 kbp, circular ExPΦ (designated ФBU01) from a vancomycin-intermediate S. aureus (VISA) strain. Assembly and annotation of ФBU01 revealed a number of putative virulence determinants encoded within a bacteriophage immune evasion cluster (IEC). Our identification of several potential ExPΦs and mobile genetic elements (MGEs) also revealed numerous putative virulence factors and antibiotic resistance genes. We describe here a previously unidentified level of genetic diversity of stealth extra-chromosomal elements in S. aureus, including phages with a larger presence outside the chromosome that likely play a prominent role in pathogenesis and strain diversity driven by horizontal gene transfer (HGT).
